IFN response in mice infected with the TURH influenza A virus strain.

<p><b>A:</b> Immunofluorescence detection of TURH influenza A virus in liver sections from Mx-IFNAR1^0/0 and Mx-IFNLR1^0/0 mice infected for 48 hours or mock-infected, using anti-influenza A virus antibody (Red) and Hoechst staining of nuclei (blue). Results are representative of two independent experiments. <b>B–C:</b> RT-qPCR analysis of influenza A virus replication in the liver (B) and lungs (C) of Mx-IFNAR1^0/0 and Mx-IFNLR1^0/0 mice. Results are expressed as influenza A virus cDNA copies per 10⁵ copies of β-actin cDNA. <b>D–E:</b> RT-qPCR analysis of <i>Oasl2</i> expression in the liver (D) and lungs (E) of Mx-IFNAR1^0/0 and Mx-IFNLR1^0/0 infected mice. Results are expressed as <i>mOasl2</i> cDNA copies per 10³ copies of β-actin cDNA. <b>F–G:</b> IFN-β (F) and IFN-λ (G) production in the liver of infected mice. Results are expressed as <i>IFN</i> cDNA copies per 10⁵ copies of β-actin cDNA. It is noteworthy that cells from IFNAR1^0/0 expressed lower basal levels of IFN, likely as a consequence of a disrupted positive feed-back loop for IFN expression in these mice.</p

Primers.

a<p>Sequence kindly provided by Professor Stephan Brand, University Hospital Munich-Grosshadern, University of Munich, Germany.</p>b<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087906#pone.0087906-OConnor1" target="_blank">[32]</a>.</p>c<p>(f) forward primer; (r) reverse primer.</p>d<p>For RT-qPCR, annealing reactions were performed at 63°C for influenza A virus and at 60°C for mOASl2, hOAS1, hMxA and β-actin.</p

Hepatocytes respond to IFN-α but not to IFN-λ.

<p><b>A:</b> Fluorescence microscopy images showing the IFN response (Mx1, green) in hepatocytes (HNF4, red), in liver sections from Mx-IFNLR1^0/0 and Mx-IFNAR1^0/0 mice infected for 48 hours with influenza A virus strain TURH. Scale bar: 15 µm. <b>B:</b> Fluorescence microscopy images showing the IFN-λ response (Mx1, green) in cholangiocytes (cytokeratin, red) in liver sections from Mx-IFNAR1^0/0 mice, mock-infected or infected for 48 hours with the TURH strain of influenza A virus or mock-infected. Scale bar: 15 µm. Results are representative of two independent experiments.</p

Release of HCV core into the supernatant of transfected cells depends on the expression of functional glycoproteins.

<p>Ten µg of <i>in vitro</i> transcribed RNA of the constructs specified below each bar were transfected into Huh-7 cells and 24 h later culture medium was harvested and filtered, whereas cells were lysed with 1% Triton X-100 in PBS. The total amount of HCV core in the cell lysate (A) and culture supernatant (B) was determined by ELISA. (C) Relative core release expressed as the fraction (in %) of total intracellular core protein that is released into the culture fluid. Mean values of two independent electroporations including the standard error of the means are given.</p

Density profiles of HCV particles derived from Con1/wt, Con1/K1846T, JFH-1 and patient serum.

<p>(A) Approximately 22 ml of filtered cell culture fluid harvested 24 h after transfection of Huh-7 cells with Con1/wt RNA were concentrated via ultracentrifugation over a 60% iodixanol cushion. In case of JFH-1 approximately 26 ml supernatant harvested 96 h post transfection and concentrated in the same manner were used. The concentrates were overlaid with a linear iodixanol gradient (0%–60%) and spun for 20 h at 110,000 g at 4°C. In a similar experiment, 0.5 ml of high titer patient serum was resolved in an iodixanol density gradient. Twelve fractions à 1ml were harvested from the top, and the amount of HCV core protein contained in each fraction was determined by Trak C ELISA, respectively. HCV core content per fraction is plotted against the density of the respective fraction. (B) Result of an analogous experiment but using culture supernatant of Huh-7 cells after transfection with Con1/wt or Con1/K1846T.</p

Impact of REMs on HCV particle release.

<p>(A) Replication of subgenomic luciferase replicons in transfected Huh-7 cells. The replication deficient replicon D318N served as negative control. Values refer to luciferase activities determined 48 h post transfection of Huh-7 cells after normalization for transfection efficiency determined 4 h post transfection. Data are taken from reference <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000475#ppat.1000475-Lohmann3" target="_blank">[17]</a>. (B) Huh-7 cells were transfected with the Con1/wt genome or Con1 genomes carrying REMs specified in the bottom of the graph. The amount of core protein released into the supernatant 24 h after transfection was determined by using core ELISA and is expressed relative to the quantity of core measured for Con1/wt. Mean values of between 2 and 11 independent repetitions (depending on the construct) including the standard deviation are given. (C) Impact of REMs in NS4B and NS3 on RNA replication as determined by intracellular accumulation of core protein (left panel). The kinetic of release of core protein into the supernatant of Huh-7 cells after transfection with the Con1/wt or the Con1/K1846T or the Con1/NS3+K1846T genome is shown in the middle panel. The right panel shows the percentage of intracellular core that is released into the culture supernatant of Huh-7 cells at various time points after transfection with each of the 3 genomes. A representative example of two independent repetitions is shown.</p

<i>In vitro</i> infectivity of Con1/wt particles released from transfected Huh7.5 cells.

<p>(A) Enhancement of HCV RNA replication by kinase inhibitor H479. Subgenomic Con1 luciferase replicons were transfected into Huh7.5 cells that were seeded into medium containing H479 at concentrations specified in the right. Cell lysates were prepared at 4 h and 48 h after transfection and luciferase activities were determined. The replication defective replicon Con1/D318N served as negative control. Cells treated with DMSO only were used as reference. For each construct, values were normalized to the luciferase activity of the respective DMSO control in order to determine the fold induction or reduction of replication. Data (mean±S.D.; n = 3) were analyzed using two-way ANOVA test. (B) Experimental approach used to detect <i>in vitro</i> infectivity of Con1 virus. (C) Immunofluorescence analysis of Huh7.5 cells 72 h after inoculation with supernatant from cells transfected with the Con1/wt genome (upper panels) or mock transfected cells (lower panels). Cells were treated either with DMSO only (Mock; left panels), or with H479 (middle panels) or with H479 and ConcanamycinA (right panels) as specified in panel (B). Cells were fixed 72 h after inoculation and NS5A was detected by immunofluorescence microscopy. (D) Detection of NS3 and NS5A expression in Huh7.5 cells inoculated with cell-free concentrated supernatant containing Con1/K1846T particles. Cells were fixed 48 h after inoculation and processed for indirect immunofluorescence. Nuclei were counterstained with DAPI.</p

Transient replication of HCV Con1-derived constructs in Huh-7 cells and release of core protein from transfected cells.

<p>(A) Schematic representation of transfected RNA genomes. The polyprotein is indicated by an open box, the individual functional proteins are separated by vertical lines. Non-translated regions are depicted as shaded bars, REMs and the position of the mutation destroying the active site of the NS5B RdRp (D318N) are specified above the respective positions in the coding region. (B) Transient RNA replication of full length Con1-derived genomes. Ten µg of <i>in vitro</i> transcribed RNA of the constructs specified in the top were transfected into Huh-7 cells that were harvested at given time points. Total cellular RNA was prepared and HCV RNA and beta-actin RNA were detected by Northern hybridization. (C) The amount of HCV RNA was determined by phospho imaging and is expressed relative to the input determined 4 h post transfection. Values were normalized for equal RNA loading as determined with the beta-actin specific signal. (D) Time course of accumulation of HCV core in cell culture supernatant of transfected Huh7 cells. Cells were transfected and seeded as described in (A). At given time points, culture medium was harvested, filtered through 0.45 µm pore-size filters, and analysed for core protein by ELISA. Duplicate measurements, mean value of duplicates and standard errors of the means are given. (E) Efficiency of core release from cells transfected with Con1/wt or the adapted genome Con1/NS3+S2197P <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000475#ppat.1000475-Bukh1" target="_blank">[27]</a>. Amounts of core protein accumulated intracellularly or in cell culture medium were determined by ELISA and used to calculate the percentage of intracellular core protein released into the supernatant of transfected cells for each given time point. Mean values of two independent electroporations including standard errors of the means are shown.</p

<i>In vivo</i> infectivity of Con1/wt, Con1/K1846T and Con1/NS3+K1846T genomes in uPA-SCID mice.

<p>Huh7-Lunet cells were transfected with either of these constructs, supernatants were collected 12 and 24 h post transfection, pooled for each construct and used for virus purification and concentration as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000475#s4" target="_blank">Materials and Methods</a>. Two mice were each inoculated with 2×10⁸ IU HCV RNA per mouse and construct (100 µl inoculum size) and viral RNA loads in sera were determined at the indicated time points after inoculation by qRT-PCR. In case of Con1/K1846T inoculated mice, one died at week 2 (not shown) and the second shortly after week 6. While sera of Con1/wt and Con1/K1846T inoculated mice contained high viral loads already in the first blood sample, Con1/NS3+K1846T-inoculated mice remained HCV RNA negative throughout the 10 weeks observation period.</p

Structural formulae of Artemisinin and synthetic derivatives belonging to the second category TVN.

<p>Structural formulae of Artemisinin and synthetic derivatives belonging to the second category TVN.</p